Abstract: In this study, Neurospora intermedia HJ 2022-06 was used for the solid-state fermentation of soybeans, following which the tempeh oil was extracted using an alkaline protease-assisted aqueous enzymatic method. A single-factor experimental design was initially used, and the extraction parameters were subsequently optimized using Box–Behnken response surface methodology. Additionally, the physicochemical properties, bioactive components, and antioxidative activities of the extracted oil were comprehensively analyzed. The optimal extraction conditions were determined using a solid-to-liquid ratio of 1:5 (g/mL), an enzyme concentration of 2.0% (w/w), and a hydrolysis time of 3.3 h. Under these conditions, the oil extraction rate reached 74.10%±1.77%, which closely matched the model-predicted value of 74.74%. Compared with the unfermented control group, fermentation with Neurospora resulted in a significant decrease in the peroxide level (P<0.01), an increase in the proportion of unsaturated fatty acids to 82.16%±0.2%, and a decrease in the ω-6/ω-3 fatty acid ratio. Furthermore, the contents of isoflavones and carotenoids increased by 33.76- and 2.05-fold, respectively. The tempeh oil also showed significant improvements in its DPPH and ABTS⁺ free radical-scavenging capacities (P<0.05). Pearson’s correlation analysis revealed strong positive correlations between the enhanced antioxidative activities and elevated carotenoid and isoflavone levels. This study presents a novel and precise strategy for producing functional oils via controlled fermentation by Neurospora.